The present invention provides polypeptides having lysophosphatidic acid acyltransferase (LPAAT) activity and polynucleotides encoding polypeptides having LPAAT activity. The present invention further provides for isolation and production of polypeptides involved in phosphatidic acid metabolism and signaling in mammalian cells, in particular, the production of purified forms of LPAAT.
LPAAT, also referred to as 1-acyl sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.51), is known to catalyze the acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) by acylating the sn-2 position of LPA with a fatty acid acyl-chain moiety. LPA and PA, while originally identified as intermediates in lipid biosynthesis (Kent, Anal Rev. Biochem. 64:315-343, 1995), have more recently been identified as phospholipid signaling molecules that affect a wide range of biological responses (McPhail et al., Proc. Natl. Acad. Sci. USA 92:7931-7935, 1995; Williger et al., J. Biol. Chem. 270:29656-29659, 1995; Moolenaar, Curr. Opin. Cell Biol. 7:203-210, 1995).
Cellular activation in monocytic and lymphoid cells is associated with rapid upregulation of synthesis of phospholipids (PL) that includes PA, diacylglycerol (DAG) and glycan phosphatidylinositol (PI). PAs are a molecularly diverse group of phospholipid second messengers coupled to cellular activation and mitogenesis (Singer et al., Exp. Opin. Invest. Drugs 3:631-643, 1994). PA can be generated through hydrolysis of phosphatidylcholine (PC) (Exton, Biochim. Biophys. Acta 1212:26-42, 1994) or glycan PI (Eardley et al., Science 251:78-81, 1991; Merida et al., DNA Cell Biol. 12:473-479, 1993), through phosphorylation of DAG by DAG kinase (Kanoh et al., Trends Biochem. Sci. 15:47-50, 1990) or through acylation of LPA at the SN2 position (Bursten et al., Am. J Physiol. 266:C1093-C1104, 1994).
Compounds that block PA generation and hence diminish lipid biosynthesis and the signal involved in cell activation are therefore of therapeutic interest in, for example, the areas of inflammation and oncology as well as obesity treatment. Therefore, compounds that block LPAAT activity have a similar therapeutic value.
The genes coding for LPAAT have been isolated in bacteria (Coleman, Mol. Gen. Genet. 232:295-303, 1992), in yeast (Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) and in plants (Brown et al., Plant Mol. Biol. 26:211-223, 1994; and Hanke et al., Eur J. Biochem. 232:806-810, 1995; Knutzon, et al., Plant Physiol. 109: 999-1006, 1995). Moreover, two human isoforms of LPAAT have been reported (West, et al., DNA Cell Biol. 6: 691-701, 1997). These isoforms are denominated LPAATxcex1 and LPAATxcex2 (West, et al., DNA Cell Biol. 6: 691-701, 1997) and are described herein. There remains, however, a need for the isolation of additional mammalian LPAATs, which can be used, for example, to screen for compounds that inhibit LPAAT activity.
The present invention provides cDNA sequences, polypeptide sequences, and transformed cells for producing isolated recombinant mammalian LPAAT. The present invention provides four polypeptides corresponding to human LPAAT isoforms. These polypeptides are designated hLPAATxcex1, hLPAATxcex2, hLPAATxcex31, hLPAATxcex32, and hLPAATxcex4. The invention further provides fragments of these polypeptides which are biologically active, i.e., which retain LPAAT activity. LPAAT activity is defined catalyzing acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA), specifically by acylating the sn-2 position of LPA with a fatty acid acyl-chain moiety.
The present invention further provides nucleic acid sequences encoding hLPAATxcex1, hLPAATxcex2, hLPAATxcex31, hLPAATxcex32, and hLPAATxcex4 and polynucleotides coding for biologically active fragments of hLPAATxcex1, hLPAATxcex2, hLPAATxcex31, hLPAATxcex32, and hLPAATxcex4. The invention further provides xe2x80x9cbiologically activexe2x80x9d polynucleotide fragments, which connotes polynucleotide fragments which encode polypeptides having LPAAT activity. The invention further provides purified LPAATs and antisense oligonucleotides for modulation of expression of the genes coding for LPAAT polypeptides. Assays for screening test compounds for their ability to inhibit LPAATs are also provided.
The present invention includes the following polynucleotides coding for hLPAATxcex1, hLPAATxcex2, hLPAATxcex31, hLPAATxcex32, and hLPAATxcex4. The invention provides the DNA sequences of: SEQ ID NO. 1 which encodes for hLPAATxcex1; SEQ ID NO. 7, which encodes hLPAATxcex2; FIG. 9, which encodes hLPAATxcex31 FIG. 10, which encodes hLPAATxcex32; and FIG. 11, which encodes and hLPAATxcex4.
The invention further includes the polypeptides for hLPAATxcex1, hLPAATxcex2, hLPAATxcex31, hLPAATxcex32, and hLPAATxcex4, specifically, the amino acid sequences of: SEQ ID NO. 2, which represents hLPAATxcex1; SEQ ID NO. 8, which represents hLPAATxcex2; FIG. 9, which represents hLPAATxcex31; FIG. 10, which represents hLPAATxcex32; and FIG. 11, which represents hLPAATxcex4.
The invention further comprises biologically active fragments of the amino acid sequences of SEQ ID NO. 2, SEQ ID NO. 8, FIG. 9, FIG. 10, and FIG. 11 or nucleotide fragments of SEQ ID NO. 1, SEQ ID NO. 7, FIG. 9, FIG. 10, and FIG. 11 which encode biologically active LPAAT. The invention further includes polynucleotides which due to the degeneracy of the genetic code encode a polypeptide of SEQ ID NO. 2, SEQ. ID NO. 8, FIG. 9, FIG. 10, and FIG. 11. The invention further includes polynucleotides capable of hybridizing to the nucleic acid sequences of SEQ ID NO. 1, SEQ ID NO. 7, FIG. 9, FIG. 10, and FIG. 11, under high stringency conditions, and which are biologically active.
Also provided by the present invention are vectors containing a DNA sequence encoding a mammalian LPAAT enzyme in operative association with an expression control sequence. Host cells, transformed with such vectors for use in producing recombinant LPAAT, are also provided with the present invention. The inventive vectors and transformed cells are employed in a process for producing recombinant mammalian LPAAT. In this process, a cell line transformed with a DNA sequence encoding LPAAT in operative association with an expression control sequence, is cultured. The claimed process may employ a number of known cells as host cells for expression of the LPAAT polypeptide, including, for example, mammalian cells, yeast cells, insect cells and bacterial cells. The present invention further provides transformed cells that expresses active mammalian LPAAT.
The present invention further provides methods for identifying compounds that increase or decrease LPAAT activity, i.e., acylation of LPA to PA. Because PA concentration is involved in numerous cellular pathways, compounds that increase or decrease acylation of LPA to PA are useful in regulating a number of cellular pathways. Such compounds can be used, for example, to augment trilineage hematopoiesis after cytoreductive therapy or to inhibit inflammation following hypoxia and reoxygenation injury (e.g., sepsis, trauma, and ARDS). Moreover, the present invention contemplates the use of such compounds in an in vitro or in vivo context.
The present invention further includes: An isolated polynucleotide encoding a polypeptide having Lysophosphatidic Acid Acyltransferase (LPAAT) activity, comprising a nucleotide sequence selected from the group consisting of:
(a) the DNA sequence of FIG. 9, FIG. 10, or FIG. 11 and biologically active fragments thereof; and
(b) a DNA sequence which encodes the polypeptide of FIG. 9, FIG. 10, or FIG. 11 and biologically active fragments thereof.
An isolated polypeptide having LPAAT activity, comprising the amino acid sequence of FIG. 9, FIG. 10, or FIG. 11 and biologically active fragments thereof.
A method for screening one or more compounds to determine whether the one or more compounds increases or decreases LPAAT activity, comprising:
(a) contacting the polypeptide of the present invention with one or more substrates for the polypeptide and with the one or more compounds; and
(b) measuring whether the LPAAT activity of the polypeptide is increased or decreased by the one or more compounds.
A method of expressing the polypeptide of the present invention, comprising:
(a) introducing into a cell a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
(i) the DNA sequence of FIG. 9, FIG. 10, or FIG. 11 and biologically active fragments thereof; and
(ii) a DNA sequence which encodes the polypeptide of FIG. 9, FIG. 10, or FIG. 11 and biologically active fragments thereof,
wherein the polynucleotide is operably linked to a promoter; and
(b) maintaining or growing said cell under conditions that result in the expression of the polypeptide.
An isolated polynucleotide encoding a polypeptide having Lysophosphatidic Acid Acyltransferase (LPAAT) activity, comprising a DNA sequence capable of hybridizing under high stringency conditions to the complement of the DNA sequences, (a) or (b), described above, and which encodes a polypeptide having LPAAT activity.